The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6

The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. We demonstrate that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/β-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient.

(J-L) Cas9 and independent sgRNAs targeting Uaf1 expressed in the posterior compartment of the wing disc using hh-Gal4 result in decreased Sens staining (green) in the posterior compartment, marked with Engrailed (En, magenta).DAPI (blue) marks nuclei.Scale bar (A-L): 50 µM (M, N) Cas9 expression driven by hh-Gal4 has no phenotype in the adult wing in the absence of sgRNAs (M), whereas concomitant expression of ebony sgRNAs results in an ebony phenotype in the posterior wing (N).Posterior, bottom.

Figure S2 .
Figure S2.Depletion of Usp46 complex components in the wing disc with independent RNAi constructs.(A-F) Independent RNAi constructs targeting Usp46 (A-C) and Wdr20 (D-F) driven by hh-Gal4 result in decreased Sens (magenta) in the posterior wing disc, marked by En (green).Dorsal, top and posterior, right.Scale bar (A-F): 20 µM

Figure S4 .
Figure S4.Usp46 complex components are expressed at invariant levels in the larval wing disc.(A-C) Usp46-V5 staining (A, magenta), Wdr20-V5 staining (B, magenta), and Uaf1-V5 staining (C, magenta) in the wing disc are observed at invariant levels.Dorsal, top.(D-L) RNAi-mediated depletion of Usp46 complex components driven by apterous (ap)-Gal4 in the dorsal compartment of the wing disc.Efficient depletion of Usp46-V5 (D-E, magenta), Wdr20-V5 (G-H, magenta) or Uaf1-V5 (J-K, magenta) is observed in the dorsal compartment, revealing specificity of the V5 signal in the ventral compartment for each Usp46 complex component.DAPI (blue) marks nuclei.Dorsal, top and posterior, right.(F, I, L) Optical zoom of the region indicated in the yellow box (E, H, K), respectively, at the dorsoventral boundary of the wing disc.There is nuclear exclusion and cytoplasmic enrichment of Usp46 (magenta, F), Wdr20 (magenta, I) and Uaf1 (magenta, L).Scale bars (A-E, G, H, J, K) and (F, I, L): 20 µM

Figure S5 .
Figure S5.Conditional CRISPR-mediated mutagenesis of Usp46 complex components results in reduced expression of the Wingless target gene senseless in the wing disc.(A-C) Cas9 and control sgRNAs targeting ebony (e) expressed in the posterior compartment of the wing disc using the hh-Gal4 (hh) driver have little effect on Sens (green) levels.The posterior of the wing disc is marked with Engrailed (En, magenta).DAPI (blue) marks nuclei.(D-L) Cas 9 and sgRNAs targeting each component of the Usp46 complex were expressed in the posterior compartment of the wing disc using the hh-Gal4 driver.CRISPR-mediated mutagenesis resulting in decreased levels of V5-tagged Usp46 (D-F), V5-tagged Wdr20 (G-I) and V5-tagged Uaf1 (J-L) is indicated by decreased V5 staining (magenta) in the posterior disc.Decreases in Sens levels (green) indicate the loss of Wingless signaling, which is observed only in the posterior compartment where Cas9 and the sgRNAs are both expressed.DAPI (blue) marks nuclei.Scale bar (A-L): 50 µM.Dorsal, top and posterior, right.(M)Quantification is shown as percentage of discs from males of each genotype with decreased Sens. N is the number of discs analyzed.Source data are provided in the Source Data file.

Figure S6 .
Figure S6.Independent sgRNAs targeting Usp46 complex components result in decreased expression of the Wingless target gene senseless.(A-I) Expression of Cas9 only (A-C), or together with independent sgRNAs targeting Usp46 (D-F) or Wdr20 (G-I) in the posterior compartment of the wing disc using the hh-Gal4 driver resulted in decreased Senseless (Sens, green) in the posterior disc.Decreased V5 staining (magenta) indicates cells with reduced Usp46 or Wdr20, respectively.DAPI (blue) marks nuclei.

Figure S7 .
Figure S7.Wingless expression is unaffected by conditional CRISPR-mediated mutagenesis of the Usp46 complex.(A-I) Cas9 and sgRNAs targeting Usp46 (A-C), Wdr20 (D-F) or Uaf1 (G-I) were expressed in the posterior compartment of the third instar larval wing disc using the hh-Gal4 driver.Reduction of Sens (green) was observed specifically in the posterior region of each wing disc examined.In contrast, Wingless (yellow) expression was unaffected.Scale bar (A-I): 50 µM

Figure S10 .
Figure S10.Arrow promotes expression of the Wingless target gene frizzled3 (fz3) in the posterior midgut.(A-C) Expression of fz3-GFP (green) in the wild-type adult posterior midgut (A, B).Quantification of fz3-GFP signal intensity as a function of distance from the midgut-hindgut boundary (MHB, yellow arrow) (C).Posterior, right.(D-F) arrow null mutant clones (magenta) in the adult intestinal epithelium.fz3-GFP expression (green).The MHB is delineated (M|H).Posterior, right.DAPI (blue) marks nuclei.(G-I) Higher magnification of white box in panel D. arrow null mutant cells displayed complete loss of fz3-GFP (white arrows).(J-L) Control clones of wild-type cells (FRT 82B, magenta) in the adult intestinal epithelium.(M-O) Higher magnification view of white box in panel J. fz3-GFP expression is not reduced in control clones (orange arrows).Scale bars (A-C) 20 µM; (D-F), (G-I), (J-L), and (M-O): 50 µM

Figure S11 .
Figure S11.Independent Usp46, Wdr20 and Uaf1 null alleles result in reduction of fz3 expression in the posterior midgut.(A-C) Usp46 MiMIC null mutant clones (magenta) in the adult intestinal epithelium.fz3-GFP expression (green).The MHB is delineated (M|H).Posterior, right.DAPI (blue) marks nuclei.(D-F) Higher magnification view of box D in panel A showing a region distant from the MHB.Usp46 MiMIC null mutant cells in this region showed complete loss of fz3-GFP (white arrows) whereas those slightly closer to the MHB showed a nearly complete reduction in fz3-GFP (yellow arrows).(G-I) Higher magnification view of box G in panel A showing region near the MHB.Usp46 MiMIC null mutant cells in this region display either a partial decrease (yellow arrows), or no decrease in fz3-GFP.Scale bars (A-C) and (D-I): 50 µM (J) Quantification is shown as percentage of clones of each genotype with decreased fz3-GFP (yellow) or the absence of fz3-GFP (gray) expression.Clones (n = number) close and far from the MHB were analyzed.* * * * p < 0.0001 (1E-6 for Usp46 MiMIC clones far from the MHB, 0 for all other genotypes, one-tailed t-test).Source data are provided in the Source Data file.

Figure S12 .
Figure S12.Wdr20 promotes expression of the Wingless target gene fz3 in the posterior midgut.(A-C) Wdr20 33 null mutant clones (magenta) in the adult intestinal epithelium.The MHB is delineated (M|H).DAPI (blue) marks nuclei.Posterior, right.(D-F) Magnification of box D from panel A showing a region distant from the MHB.The relative decrease in fz3-GFP correlates with distance of the Wdr20 33 mutant clone from the MHB.Wdr20 33 mutant clones (magenta, white arrows) in this region display complete loss of fz3-GFP.(G-I) Magnification of box G from panel A showing region near MHB.Wdr20 33 mutant clones located closer to the MHB display a partial reduction in fz3-GFP (yellow arrows).A few clones display an almost complete loss of fz3-GFP (white arrows).Scale bars (A-C) and (D-I): 50 µM.

Figure S13 .
Figure S13.Inactivation of Uaf1 in the posterior midgut results in decreased expression of the Wingless target gene fz3.(A-C) Uaf1 3 null mutant clones (magenta) in the adult intestinal epithelium.The MHB is delineated (M|H).DAPI (blue) marks nuclei.Posterior, right.(D-F) Magnification of box D from panel A showing a region distant from the MHB.Uaf1 3 mutant clones (magenta) in this region display complete loss of fz3-GFP (white arrows) or strong reduction of fz3-GFP (yellow arrows).(G-I) Magnification of box G from panel A showing region near MHB.Uaf1 3 null mutant clones located in this region display a partial reduction in fz3-GFP (yellow arrows).Scale bars (A-C) and (D-I): 50 µM.

Figure S14 .
Figure S14.Independent Wdr20 mutant reduces fz3 expression in the posterior midgut.(A-C) Wdr20 34 null mutant clones (magenta) in the adult intestinal epithelium.fz3-GFP expression (green).DAPI (blue) marks the nuclei.The MHB is delineated (M|H).Posterior, right.(D-F) Magnification of box D from panel A showing a region distant from the MHB.Wdr20 34 mutant clones (magenta) display complete loss of fz3-GFP (white arrows) or strong reduction in in fz3-GFP (yellow arrows).(G-I) Magnification of box G from panel A showing Wdr20 34 clones (magenta) closer to the MHB, which display a partial reduction in fz3-GFP (yellow arrows).Scale bars: (A-C) and (D-I): 50 µM

Figure S16 .
Figure S16.Loss of the Usp46 complex does not block the asymmetric division of ISCs.(A-H) The enteroblast-specific transcriptional marker Su(H)-lacZ (red) marks progenitor cells derived from the asymmetric division of intestinal stem cells.Midguts from Usp46, Wdr20 or Uaf1 null allele transheterozygotes displayed an increased number of enteroblasts when compared to wild-type midguts.Panels B, D, F, and H are higher magnification views of panels A, C, E, and G, respectively.DAPI (blue) marks the nuclei.Scale bars (A,C,E,G) and (B,D,F,H): 20 µM (I) Quantification of Su(H)-lacZ positive cells is shown as mean (red line).0.051mm 2 fields in either the R5 region adjacent the MHB or at the R4/R5 boundary were analyzed.Each point represents an individual posterior midgut.* * * * p < 0.0001 (p values in order: 3.4E-5, 2.9E-7, 1E-8, <1E-8, 4.8E-7, 5.67E-6, 1E-7 and 8.4E-6, two tailed t-test).Source data are provided in the Source Data file.

Figure S17 .
Figure S17.Usp46 and Wdr20 prevent non-autonomous overproliferation of neighboring ISCs.(A, B) Control clones (FRT82B) of wild-type (WT) cells marked with GFP (green) were generated using the MARCM system in the wild-type adult midgut epithelium.esg-lacZ (magenta).Panel B is a higher magnification view of panel A. (C-F) Usp46 and Wdr20 null mutant clones (marked with GFP, green) induce non-autonomous regional increases in the number of intestinal stem and progenitor cells (marked by esg-lacZ, magenta).Higher magnification views of panels C and E are shown in panels D and F, respectively.Scale bars (A, C, E) and (B, D, F): 20 µM (G) Quantification of esg-lacZ positive cells is shown as mean (red line).Each point represents an individual posterior midgut.0.051mm 2 fields in the R5 region were analyzed.* * * * p < 0.0001 (5E-8 for Usp46 1 and <1E-8 for all other genotypes, two-tailed t-test).Source data are provided in the Source Data file.

Figure S18 .
Figure S18.Uaf1 prevents the non-autonomous overproliferation of neighboring ISCs.(A-F) Control clones (FRT42D) of wild-type (WT) cells marked with GFP (green) were generated using the MARCM system in the wild-type adult midgut epithelium.Delta-lacZ (red) marks intestinal stem cells and DAPI (blue) marks the nuclei.Panels D, E, and F are higher magnification views of the boxed region in panels A, B and C, respectively.(G-L) Uaf1 4 null mutant clones (marked with GFP, green) induce the non-autonomous regional overproliferation of intestinal stem cells (marked by Delta-lacZ, red).DAPI (blue) marks the nuclei.Panels J, K, and L are higher magnification views of the boxed area in panels G, H, and I, respectively.Scale bars (A-C, G-I) and (D-F, J-L): 20 µM (M) Quantification of Delta-lacZ positive cells is shown as mean (red line).Each point represents an individual posterior midgut.0.051mm 2 fields in the R5 region were analyzed.****p < 0.0001 (4.1E-7 and 8E-8, two-tailed t-test).Source data are provided in the Source Data file.

Figure S20 .
Figure S20.The Usp46 complex acts upstream of the destruction complex.(A-C) Expression of the Wingless target gene reporter naked-lacZ (nkd-lacZ, magenta) in a wild-type third instar larval wing disc.nkd-lacZ expression is observed in a broad band surrounding the dorsoventral boundary.DAPI (blue) marks the nuclei.Dorsal, top.(D-F) Axin null mutant clones (marked by the absence of GFP, green) in the wing disc display ectopic expression of nkd-lacZ (magenta), indicating the constitutive activation of Wingless signaling.(G-L) Usp46 1 Axin (G-L) or Wdr20 33 Axin (J-L) double null mutant wing disc clones (marked by absence of GFP, green) display ectopic expression of nkd-lacZ (magenta).Scale bars: (A-C) and (D-L): 50 µM

Figure S22 .
Figure S22.Usp46 complex stability.(A) Auto-stabilization of the Usp46 complex.Drosophila S2R+ cells were transfected with individual or combinations of HA-tagged Usp46 complex components.Usp46 is stabilized upon co-transfection of Wdr20 and to a lesser degree upon co-transfection of Uaf1.Co-transfection of Usp46 stabilizes Uaf1 and Wdr20.Tubulin was used as a loading control.(B) Knockdown of Usp46 complex components resulted in decreased Arrow levels.RNAi-mediated knockdown of the Usp46 complex decreases the steady-state levels of Arrow.S2R+ cells were treated with Ctrl or Usp46 complex dsRNAs as indicated, followed by immunoblotting with Arrow antibody.Tubulin was used as a loading control.(C) Quantitation of Arrow levels normalized to tubulin.Mean and standard deviations are shown, n=3.*p = 0.0018 for Usp46 + Uaf1,**p = 0.0231for Wdr20 + Uaf1 (two tailed t-test).Source data are provided in the Source Data file.

Figure S23 .
Figure S23.Wdr20 and Uaf1 stabilize Usp46 in larval wing imaginal discs.(A-C) hh-Gal4-driven expression of a control RNAi construct targeting the yellow (y) gene.No reduction of Usp46-V5 (green) was observed.(D-I) hh-Gal4-driven expression of two independent RNAi constructs targeting Wdr20 result in decreased Usp46-V5 in the posterior wing disc.(J-L) hh-Gal4-driven expression of an RNAi construct targeting Uaf1 results in decreased Usp46-V5 in the posterior wing disc.Scale bar (A-L): 50 µM (M) Quantification of percentage of wing discs with reduction of Usp46-V5 upon expression of the indicated RNAi construct.****p < 0.0001 (0 for all genotypes, one-tailed t-test).Source data are provided in the Source Data file.

Figure S24 .
Figure S24.Usp46 and Wdr20 inactivation does not disrupt membrane integrity or cause cell death.(A-C) Usp46 1 null mutant clones (magenta) in the adult posterior midgut.Arrow (green) levels are decreased at the cell membrane and in the cytoplasm.Dlg1 (cyan) localization at the cell membrane and DAPI (blue) in the nuclei are normal, indicating normal apico-basal polarity.(D-F) Wdr20 33 null mutant clones (magenta) displayed wild-type staining of Dlg1 (cyan) and DAPI (blue), while showing reduction in Arrow levels (green).Scale bar (A-F): 10 µM

Supplementary Table 1. Usp46 complex mutant alleles
*All positions with respect to the transcriptional start site